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Phase I: Two Dimensional gel Electrophoresis
Phase II: 2D Protein gel analysis tool
Internship details
Where: Department of Bioengineering (GWC 643)
As: Research Assistant
Employer: Dr. Padmini Komalavilas
Requirements: Proteomics wet-lab hands-on experience and Bioinformatics programming skills in Matlab.
2D protein gel analysis tool for studying the protein phosphorylation changes associated with contraction and relaxation of vascular
smooth muscle
Phase I
Introduction
Goals
Methodology and Analysis
Collecting and dissecting tissue to obtain coronary artery Treatment of coronary artery with drugsTissue PulverizationProtein QuantificationProtein SeparationStainingScanning
Results
Presentation Outline
Phase IIIntroduction
Goals
Methodology and Analysis
Counting number of proteins on gel imagesDisplaying location and intensity of proteinsComparing Control gel image with treated gel images
Demonstration of Application
Results
Presentation Outline
Discussion
Conclusions and Future Work
Acknowledgments
References
Phase I: Introduction
Proteome: Proteome can be defined as the complete set of proteins that ultimately results from genome transcription in a given cell, tissue or organism.
Complexity of Proteome is far beyond genome
Proteomics: Proteomics can be defined as the qualitative and quantitative comparison of proteomes under different conditions to further unravel biological processes.
Phase I: Introduction
Protein phosphorylation: The chemical addition of a phosphate group (phosphate and oxygen) to a protein.
Protein phosphorylation is an important post-translational modification since it results in cellular activity and is also reversible.
Phase I: Introduction
Smooth muscle cells are long (15 μm to 500 μm) and spindle-shaped with elongated nuclei. Smooth muscle is found in the walls of many hollow organs. In a smooth muscle cell the nucleus is centrally located and that smooth muscle is more eosinophilic
Yellow Arrow - Nuclei of Smooth Muscle CellGreen Arrow - Width of Smooth Muscle Tissue
Phase I: Goals
To study protein phosphorylation changes associated with the contraction and relaxation of vascular smooth muscle using two dimensional gel electrophoresis.
To study total proteins expressed during contraction and relaxation of vascular smooth muscle using spyro ruby stain.
To study phosphorylated proteins expressed during contraction and relaxation of vascular smooth muscle using phospho protein stain.
Phase I: Methodology and Analysis
Tissue acquisition
Dissecting the tissue to obtain coronary artery
1 = pulmonary trunk; 2 = Left anterior descending coronary artery; 3 = right coronary artery with its right marginal branch.
Phase I: Methodology and Analysis
Tissue that is not treated with drugs is called as control.
Treatment 1: Contraction is induced by treating the tissue with 1uM serotonin for 10 min.
Treatment 2: Smooth muscle tissue is treated with 1uM serotonin for 5 min followed by 10uM sodium nitroprusside for 5 min to induce relaxation.
Treatment 3: Tissue is treated with 1uM serotonin followed by 100uM papaverine for 5 min to induce relaxation.
Drug Serotonin:
The chemical name for serotonin is 5-hydoxytryptamine which is often abbreviated to 5-HT.One role of this 'wonder drug' is as a neurotransmitter, allowing numerous functions in the human body which includes muscle contraction.
Phase I: Methodology and Analysis
Drug Papaverine
Drug Sodium Nitroprusside
Papaverine is used to improve blood flow. It works by relaxing the blood vessels so that blood can flow more easily to the heart and through the body.
It acts by relaxation of vascular smooth muscle. Sodium nitroprusside breaks down in the blood and releases a chemical call nitric oxide (NO). Nitric oxide enters the muscle cells in the walls of the blood vessels and causes them to relax.
Tissue pulverization: After treatment with drugs tissue is frozen in liquid nitrogen and pulverized using mortar and pestle.
The tissue is solubilized in urea, dithiothreitol, CHAPS (UDC) buffer by vortexing for 30 min at room temperature. Extracted proteins are separated by centrifuging the extract at 14,000 rpm for 10 min at 4oC using a centrifuge. Supernatent are transferred to a fresh tube.
Phase I: Methodology and Analysis
Protein Quantification:
Proteins are quantitated using Bradford protein assay. The method is based on the proportional binding of the dye Coomassie to proteins and measuring the absorbance of the complex in a spectrophotometer and compared to known amount of standard proteins.
Phase I: Methodology and Analysis
Protein Quantification:
750750200 200 ulul50ul50ul1010’’
750750200 200 ulul50ul50ul1010
750750210 210 ulul40ul40ul88’’
750750210 210 ulul40ul40ul88
750750220 220 ulul30ul30ul66’’
750750220 220 ulul30ul30ul66
750750230 230 ulul20ul20ul44’’
750750230 230 ulul20ul20ul44
750750240 240 ulul10ul10ul22’’
750750240 240 ulul10ul10ul22
750750250 250 ulul0ul0ul00’’
750750250 250 ulul0ul0ul00
VolVol Assay Assay mixmix
VolVol H2OH2OVolVol/ / StandardStandard
7507502492491ul1ul5HT + Papa 25HT + Papa 2
7507502492491ul1ul5HT + Papa 15HT + Papa 1
7507502492491ul1ul5HT + SNP 25HT + SNP 2
7507502492491ul1ul5HT + SNP 15HT + SNP 1
7507502492491ul1ul5HT 25HT 2
7507502492491ul1ul5HT 15HT 1
7507502492491ul1ulControl 2Control 2
7507502492491ul1ulControl 1Control 1
VolVol Assay MixAssay MixVolVol H2OH2OVolVol of Proteinof ProteinProteinProtein
Phase I: Methodology and Analysis
Phase I: Methodology and Analysis
Protein Quantification:
231.2231.218.818.85.3 5.3 ug/ulug/ul5HT + 5HT + PapaverinePapaverine
233.1233.116.916.95.9 5.9 ug/ulug/ul5HT + SNP5HT + SNP
236.9236.913.113.17.63 7.63 ug/ulug/ul5HT5HT
225.6225.624.424.44.1 4.1 ug/ulug/ulControlControl
UDC bufferUDC buffer100ug100ug
Protein Separation Two-dimensional polyacrylamide gel electrophoresis
Key principles
• Proteins differ from each other in terms of their mass and charge.Both these properties can be used to separate proteins by gel electrophoresis.
• The successive application of both techniques in perpendicular directions (two dimensions) provides maximum separation and allows thousands of proteins to be resolved.
• Staining the gel reveals the positions of individual proteins as spots or smudges.
Phase I: Methodology and Analysis
1st dimension Separation by charge(isoelectric focussing)
2nd dimension Separation by molecular weight
(SDS-PAGE)kDa
pH 3 pH 10
pI
Phase I: Methodology and Analysis
Protein Separation Two-dimensional polyacrylamide gel electrophoresis
First Dimension Methodology of a 2D Gel
It uses a procedure called isoelectric focusing, which separates polypeptide chains depending on the surrounding pH and the charge of the protein (negative or positive).
Phase I: Methodology and Analysis
Equilibration Solutions
10mL Stock Re-equilibration Buffer + 250mg iodacetamide
Solution 210mL Stock Re-equilibration Buffer + 100mg DTTSolution 1
Strip Equilibration
Phase I: Methodology and Analysis
Second Dimension
The second dimension is usually an SDS-polyacrylamide gel electrophoresis, which allows the separation of the proteins based on their molecular size
Phase I: Methodology and Analysis
StainingPro-Q® Diamond phosphoprotein gel stain
Staining procedure1 Fix the gel2 Wash the gel 3 Stain the gel 4 Destain the gel 5 Wash the gel
Imaging and Documenting the Gel
Phase I: Methodology and Analysis
StainingSYPRO Ruby protein gel stain
Staining procedure1 Fix the gel2 Wash the gel 3 Stain the gel 4 Destain the gel 5 Wash the gel
Imaging and Documenting the Gel
Phase I: Methodology and Analysis
Phase II: 2D Protein Analysis tool
To develop a 2D Protein analysis tool to obtain total count of proteins along with the location and intensity of proteins.
To compare two two-dimensional (2-D) protein gels by location of proteins using 2D protein analysis tool.
0 50 100 150 200 250 3000
100
200
300
400
500
600
700
800
900
50 100 150 200 250 3000
100
200
300
400
500
600
700
800
900
Phase II: Methodology
Before Thresholding After Thresholding
Counting number of proteins
Fastcentroid function returns the centroid coordinates for the connected components (iind, jind) and the number of pixels of each component (numberofpixels). This software was developed by Marcelino Sanchez Gonzalez in May 2002.
Phase II: Methodology
Finding similar proteins in control and treated gel images
The matching criterion is based on the Euclidean distances. The straight line distance between two points
Phase II: Methodology
411195HT + Papaverine
719235HT + SNP
513215HT
71827Control
Common proteinsPhosphoproteinSpyro ruby
0
5
10
15
20
25
30
Spyro ruby 27 21 23 19
Phosphoprotein 18 13 19 11
Common proteins 7 5 7 4
Control 5HT 5HT + SNP 5HT + Papaverine
Phase II: Results
95HT + Papaverine5HT + SNP
65HT + Papaverine5HT
85HT + SNP5HT
95HT + PapaverineControl
125HT + SNPControl
115HTControl
Common proteins
Spyro ruby stain
0
2
4
6
8
10
12
Spyro ruby stain
Common proteins 11 12 9 8 6 9
5HT 5HT + SNP 5HT + Papav5HT + SNP 5HT + Papav5HT + Papav
Control Control Control 5HT 5HT 5HT + SNP
Phase II: Results
125HT + Papaverine5HT + SNP
65HT + Papaverine5HT
85HT + SNP5HT
125HT + PapaverineControl
105HT + SNPControl
135HtControl
Common proteins
Phosphoproteinstain
0
2
4
6
8
10
12
14
Phosphoprotein stain
Common proteins 13 10 12 8 6 12
5Ht 5HT + SNP 5HT + Papav 5HT + SNP 5HT + Papav 5HT + Papav
Control Control Control 5HT 5HT 5HT + SNP
Phase II: Results
There is minimal change in the total number of proteins picked up by spyroruby stain during control, contraction and relaxation of vascular smooth muscle.
There is decrease in the total number of phosphorylated proteins picked up by phosphor protein stain during control, contraction and relaxation of vascular smooth muscle when compared to the total number of proteins.
Conclusion
Discussion
Limitations of 2-D gel electrophoresis.
Limitations of 2-D Protein gel analysis tool
Significance of the project
Future work
To develop second version of 2D protein gel analysis tool which can match proteins based on the intensity
To study phosphorylated proteins by using anti-phosphoamino acid antibodies.
Referenceshttp://arxiv.org/ftp/cs/papers/0305/0305048.pdfhttp://www.reindeergraphics.com/tutorial/index.shtmlhttp://www.csse.uwa.edu.au/~pk/Research/MatlabFns/Match/matchbycorrelation.mhttp://www.csse.uwa.edu.au/~pk/Research/MatlabFns/
Acknowledgments
I would like to express my sincere gratitude to Dr. PadminiKomalavilas for suggesting the project and providing valuableGuidance.
Many thanks to
Dr. Lokesh Joshi
Dr. Kirkmann Liff
Mr. Ashutosh Kulkarni
Mr. Vinod Swarna
0.8980.8981010’’1212
0.9000.90010101111
0.8280.82888’’1010
0.8280.8288899
0.7600.76066’’88
0.7450.7456677
0.6750.67544’’66
0.6730.6734455
0.5720.57222’’44
0.5730.5732233
0.4360.43600’’22
0.4500.4500011
BlankBlank
Net ANet AUgUgIDID
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 0’ 2 2’ 4 4’ 6 6’ 8 8’ 10 10’
Series1
Phase I: Methodology and Analysis
0.7420.7425HT + Papa 25HT + Papa 2
0.7390.7395HT + Papa 15HT + Papa 1
0.7840.7845HT + SNP 25HT + SNP 2
0.7730.7735Ht + SNP 15Ht + SNP 1
0.8800.8805HT 25HT 2
0.8610.8615HT 15HT 1
0.6660.666Control 2Control 2
0.6840.684Control 1Control 1
231.2231.218.818.85.3 5.3 ug/ulug/ul5HT + Papa5HT + Papa
233.1233.116.916.95.9 5.9 ug/ulug/ul5HT + SNP5HT + SNP
236.9236.913.113.17.63 7.63 ug/ulug/ul5HT5HT
225.6225.624.424.44.1 4.1 ug/ulug/ulControlControl
UDC bufferUDC buffer100ug100ug
Phase I: Methodology and Analysis